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6th-8th November 2017 University of Leiden, The Netherlands

“SYNTHETIC METHODS FOR GLYCAN ASSEMBLING”

PROCEEDINGS

THIRD WORKSHOP

INTRODUCTION

GLYCOVAX - A Training Network for the Rational Design of the Next Generation of Well-Defined Glycoconjugate Vaccines

GLYCOVAX is a European Training Network (ETN) funded in the framework of H2020 MarieSkłodowska- Curie ITN programme. The GLYCOVAX network aims at the education of promisingyoung scientists who will learn how to rationally design well-defined and innovative glycoconjugate vaccines to improve current preventive therapies and tackle unmet medicalneeds. The project is based on a profound interaction between the academic and industrial sectors, involving 8 academic groups and 2 industrial partners. In this highly multidisciplinaryenvironment 14 Early Stage Researchers (ESRs) are trained in the growing field of glycoscienceand vaccinology, enriching their skills and combining different state-of-the-art methodologies forthe rational design of innovative glycoconjugates.

For more details and news, visit the website www.glycovax.eu.

WORKSHOPS

Six workshops will be organized during GLYCOVAX. In these workshops, the ESRs will attendseminars from internationally recognized experts in scientific areas related to the network objectives. The topics selected for the workshops aim at covering the state-of-the-art of disciplines strongly connected with the GLYCOVAX program. These forums are intended also tostimulate the establishment of new scientific collaborations inside and outside the network.

In particular, the third Workshop “Synthetic methods for glycan assembling” has been organized Universiteit Leiden, Leiden, The Netherlands. Lectures dealt with:

• Glycosylation reaction. • Protection and deprotection strategies.• Approaches for the oligosaccharide assembly. • Automated synthesis. • One pot, chemoselective, iterative syntheses. • Chemoenzymatic synthesis

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WORKSHOP: SYNTHETIC METHODS FOR GLYCAN ASSEMBLINGMonday 6th November 201708.30 - 09.00 Arrival and registration

09.00 - 09.15 Welcome SessionJeroen Codee - Universiteit Leiden, The Netherlands

09.15 - 10.15 Fundamentals of Synthetic Carbohydrate ChemistryJeroen Codee - Universiteit Leiden, The Netherlands

10.15 - 10.30 Coffee break

10.30 - 11.30 Strategies in oligosaccharide synthesisJeroen Codee - Universiteit Leiden, The Netherlands

11.30 - 12.30 Chemoenzymatic Synthesis of OligosaccharidesIvan Gagarinov - Universiteit Utrecht, The Netherlands

12.30 - 14.00 Lunch

14.00 - 15.00 Chemical tools to study and modulate vaccine processingSander van Kasteren - Universiteit Leiden, The Netherlands

15.00 - 15.15 Coffee break

15.15 - 16.15 Gold-nanoparticles as tool to interrogate and investigate conjugated vaccinesFabrizio Chiodo - Universiteit Leiden, The Netherlands

16.15 - 17.15 Immunogenic glycans at the host-parasite interfaceRon Hokke - Universiteit Leiden, The Netherlands

19.30 Networking dinner (Restaurant City Hall, Stadhuisplein 3)

TRAINING EVENT III 6th-10th November 2017 - University of Leiden, The Netherlands

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TRAINING EVENT III 6th-10th November 2017 - University of Leiden, The Netherlands

WORKSHOP: SYNTHETIC METHODS FOR GLYCAN ASSEMBLINGWednesday 8th November 201709.30 - 10.30 Activity-based glycosidase profiling

Hermen Overkleef - Universiteit Leiden, The Netherlands

10.30 - 10.45 Coffee break

10.45 - 11.45 Glycolipids in health and diseaseHans Aerts - Universiteit Leiden, The Netherlands

11.45 - 12.45 Synthesis of ADP-ribosylated biomoleculesDima Filippov - Universiteit Leiden, The Netherlands

12.45 Lunch + Team Building activity

FUNDAMENTALS OF SYNTHETIC CARBOHYDRATE CHEMISTRYJeroen CodéeUniversiteit Leiden, Leiden, The Netherlands

Although tremendous progress has been made over the last decades in the assembly of oligosaccharides and glycoconjugates, no general solution exists for the formation of a glycosidiclinkage. This is the result of the huge variation in types of glycosidic linkages that can be formed between two glycosides as a consequence of the many regio- and stereoisomers that can generated.The structural diversity is further expanded by the many different protecting and functional groups thatare present in the building blocks. This lecture presents the state-of-the-art in the filed of oligosaccharidesynthesis and introduces the most recent insights into how the stereoelectronic effects of the ringsubstituents affect the reactivity of carbohydrate building blocks and control the overall outcome of aglycosylation reaction.

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STRATEGIES IN OLIGOSACCHARIDE SYNTHESISJeroen CodéeUniversiteit Leiden, Leiden, The Netherlands

By the hand of selected examples different state-of-the-art strategies in oligosaccharide assembly will be introduced. Special attention will be paid to the construction of complex bacterial oligosaccharidesusing both solution phase and automated solid phase synthesis methods. Building on recent insights into structure-reactivity relationships of carbohydrate building blocks the stereoselective construction of glycosidic linkages will be described.

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CHEMOENZYMATIC SYNTHESIS OF OLIGOSACCHARIDESIvan GagarinovUniversiteit Utrecht, Utrecht, The Netherlands

It has been well established that a chemoenzymatic approach, which combines in itself artistic flexibilityof chemical synthesis and precision of glycosyltransferases, is a powerful method to obtain complex carbohydrates. This hour will serve as an introduction to the most commonly performed enzymatic reactions with carbohydrates. Topics of enzymatic sialylation, galactosylation, GlcNAc-ylation and fucosylation will be discussed. Particular attention will be devoted to practical aspects of enzymatic synthesis i.e. a general algorithm of reaction set up, purification, and characterization of newly synthesized compounds. Some practical strategies to access some rare sugar nucleotides will also bementioned. Finally, important approaches to asymmetry in chemoenzymatic synthesis will be discussedwith examples from recent publications within our group and those of others.

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BIOORTHOGONAL CHEMISTRY TO STUDY ANTIGEN PROTEOLYSIS AND PRESENTATIONSander van KasterenUniversiteit Leiden, Leiden, The Netherlands

Glycans can have drastic roles on the rates and amount by which antigens are presented. Studying thepresentation of any antigenic peptides in MHC class I and II is challenging. Any modification of the antigen to make it visible alters its physiochemical properties and thereby its intracellular fate of the antigen. For example, the linkage of fluorophores to antigen alter its processing for MHC loading andcan dramatically change its presentation. Fluorophores can alter the uptake mechanism by the cells, disrupt protease cleavage sites, and affect their transport through channels involved in antigen processing, such as Sec61b and the TAP-transporter. Therefore, large pendant modifications, such asprotein reporters or fluorophores, do not provide a genuine picture of how naïve antigen is processedby antigen presenting cells (and can even enhance antigen presentation).We have recently deployed bioorthogonal chemistry to this problem to circumvent two major issues inantigen presentation. Firstly, we have used substitutions with isosteric bioorthogonal variants (e.g. anazidohomoalanine or propargylglycine to replace isosteric Methionine or Isoleucine) to image antigen(and pathogen) uptake and presentation, especially using correlative light-electron microscopy. Secondly, using bioorthogonal protection/deprotection strategy, we are beginning to address the question of how fast these processes occur – from uptake through degradation and presentation; aswell as that of subsequent T-cell activation.

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GOLD-NANOPARTICLES AS TOOL TO INTERROGATE AND INVESTIGATE CONJUGATED VACCINESFabrizio ChiodoUniversiteit Leiden, Leiden, The Netherlands

Glycan-based conjugate vaccines have been successfully used to protect society against differentbacteria but the evolutionary pressure requires always new tools to design and prepare new and efficient vaccines. Different questions are still open in the field: what is the mechanism of action of theseconjugated vaccines? How can we improve antibody titers and quality? Can we design better vaccinesdecreasing doses and recall injections?In trying to answer some of these questions, chemistry and its tools must play a key role.During this lecture, the design and preparation of carbohydrate-coated gold-nanoparticles will be described as tools to interrogate and investigate conjugated vaccines.

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IMMUNOGENIC GLYCANS AT THE HOST PARASITE-INTERFACECornelis HokkeUniversiteit Leiden, Leiden, The Netherlands

Glycoprotein antigens of parasitic helminths play important roles in activating and shaping immune responses of the host. Some types of glycans give rise to anti-glycan antibody responses of which it is yetunclear how they are related to resistance or susceptibility to specific helminth infections. Other types of glycans interact with lectins and pattern recognition receptors of the host thereby stimulating or modulatingimmune responses to the parasite. Therefore, helminth protein glycosylation, which is often species- andprotein-specific and developmentally regulated, should be considered as an important factor in vaccine development. On the one hand antigenic glycans may be vaccine targets themselves, and on the otherhand the glycans influence immune responses to the protein vaccine candidate by triggering C-type lectinsand modulating antigen processing. I will summarise our knowledge on helminth glycan structure andassociated immunology, and I will discuss consequences and considerations for the development and production of glycoprotein/glycoconjugate vaccine candidates.

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ACTIVITY-BASED GLYCOSIDASE PROFILINGHerman OverkleeftUniversiteit Leiden, Leiden, The Netherlands

Activity-based protein profiling (ABPP) is a rapidly emerging field in chemical biology research. Enzymes that employ a mechanism in processing their substrate that involves formation of a covalent enzyme-intermediate adduct can be blocked by mechanism-based suicide inhibitors: compounds thatreact within the enzyme active site to form a covalent and irreversible adduct. Introduction of a reportermoiety (‘TAG’ in the below picture) yields an activity-based probe (ABP) through which enzyme activities can be discovered (comparative ABPP) and the efficacy enzyme inhibitors in complex biological systems analyzed (competitive ABPP).Our work on ABPP development focuses on retaining glycosidases: hydrolytic enzymes able to cleaveinterglycosidic linkages and that do so through the formation of covalent enzyme-substrate intermediates. Configurational and functional analogues of the natural product and mechanism-basedretaining beta-glucosidase inhibitor, cyclophellitol, prove to be highly versatile tools to study retainingglycosidases of various nature and origin in relation to human health and disease, but also in the fieldof biotechnology. In this lecture the current state in the design, synthesis and application of synthetic cyclophellitol derivatives in studying retaining glycosidases will be presented. Discussed subjects willinclude 1) diagnosis of human lysosomal exoglycosidases in relation to lysosomal storage disorders; 2)glycosylation of cyclophellitol derivatives to arrive at retaining endoglycosidase ABPs and 3) application of glycosidase ABPs in the functional profiling of fungal secretomes for the discovery of glycosidases for biotechnology application.

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GLYCOLIPIDS IN HEALTH AND DISEASE Hans AertsUniversiteit Leiden, Leiden, The Netherlands

The lecture will deal with glycosphingolipids in health and disease. The unique class of lipids is introduced by a description of their biosynthesis and degradation. Acquired and inherited diseases inmetabolism of glycosphingolipids are discussed. Attention is focussed to new insights in pathophysiological mechanisms, therapy approaches as well as biomarkers. The lecture will highlightthe successful translation of fundamental knowledge to clinical care.

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SYNTHESIS OF ADP-RIBOSYLATED BIOMOLECULESDmitri FilippovUniversiteit Leiden, Leiden, The Netherlands

Given an important role ADP-ribosylation plays in various cellular processes, detailed understanding ofits structure and function will greatly enrich our view of living organisms. For structural and functionalstudies on ADP-ribosylation it is desirable to have access to well-defined chemically prepared modifiedbiomolecules such as ADPr-oligomers, ADPr-amino acids, ADPr-peptides and ADPr-oligonucleotides.ùHowever, synthetic methodologies that would enable preparation of ADP-ribose derivative of variousnature are scarce. Our laboratory has set out to improve the availability of molecular tools in ADP-ribosylation field and over the years we have developed synthetic routes towards ADPr-peptidesand linear oligo-ADPr-chains. Much more has still to be done to address the current limitations of chemical ADP-ribosylation. I would like to discuss our recent advances in the synthesis of ADPr-peptides, both native ones and thosecontaining stabilized analogues of ADPr-amino acid residues, such as carba-ADPr-peptides. In addition,I would like to address the synthetic challenges in the synthesis of branched oligo-ADPr-chains.

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Organization andGraphic project by:This project has received funding from the European Union’s Horizon 2020

research and innovation programme under the Marie Skłodowska-Curie grant agreement No 675671